高级生物化学与分子生物学专题肿瘤功能基因组学.pptxVIP

会计学; DNA Recombination Technology ; DNA recombination technology ; 1 DNA cloning 2 tool enzyme 3 target gene 4 gene vector;1 DNA cloning ? It is a process of DNA molecular amplification. ? Usually, the first a target DNA fragment is inserted to a vector and a recombinant (replicon) is constructed. ? The second the recombinant is transformed into host cell and screen out the cell containing the recombinant. ? The last that cell is amplified, namely a mass of target DNA molecule is gained.;2 tool enzyme ? restriction endonuclease ? DNA ligase ? DNA polymerase I ? reverse transcriptase ? polynucleotide kinase ? end-transferase ? alkaline phosphatase;structural character of cutting site recognized by restriction enzyme;3 target gene The interested gene is the target gene; source of the target gene * It is from genomic DNA directly, this is prokaryotic gene only generally. * It is from artificial synthesis, this is simple polypeptide gene generally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).; synthesize cDNA from mRNA;genomic library;5;4 gene vector ? The gene vectors are DNA molecules, which structure is reconstructed. ? They can carry target DNA fragment ? The target gene or DNA fragment is amplified and expressed. ; vector;;;the procedure of gene cloning;;cut and ligate target gene and vector; recombinant transformation ; recombinant screening; target gene amplification;;;;;重组子pGLP-1431~152 XhoI和BglII双酶切后，琼脂糖凝胶电泳鉴定结果; target gene expression;Expression analysis of four expression vectors in by SDS ;;; DNA Recombination Technology ;;; RNA Analysis Northern Blot : expressed genes RT-PCR: expressed genes In situ hybridization: gene location cDNA microarray : differentially expressed genes Bioinformatics ;Protein Analysis Western Blot: protein concentration Immunohistochemical Technique: protein location 2-D Electrophoresis: